Journal of Infectious Diseases Advance Access published April 29, 2014 1 Suppression of basophil histamine‐release and other IgE‐dependent responses in childhood Schistosoma mansoni/hookworm co‐infection
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Angela Pinot de Moira1, Colin M. Fitzsimmons1, Frances M. Jones1, Shona Wilson1, Pierre Cahen1, Edridah Tukahebwa2, Harriet Mpairwe3, Joseph K. Mwatha4, Jeffrey M. Bethony5, Per S. Skov6, Narcis B. Kabatereine2, and David W. Dunne1
2
Vector Control Division, Ministry of Health, Kampala, PO Box 1661, Uganda
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MRC/UVRI Uganda Research Unit on AIDS, P.O. Box 49, Entebbe, Uganda
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Centre for Biotechnology Research and Development, Kenya Medical Research Institute, Nairobi,
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Department of Pathology, University of Cambridge, Cambridge, CB2 1QP, United Kingdom
P.O. BOX 54840 – 00200, Kenya
Department of Microbiology, Immunology, and Tropical Medicine, George Washington University,
Washington, DC 20037, USA 6
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RefLab ApS, DK‐2200 Copenhagen, Denmark
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Address all reprint requests or correspondence to: Angela Pinot de Moira, PhD, Department of Pathology, University of Cambridge, Cambridge, United Kingdom CB2 1QP, Tel: +441223 333338; fax: +441223 333741; Email: [email protected]
Alternative corresponding author: Prof. David Dunne, PhD, Department of Pathology, University of Cambridge, Cambridge, United Kingdom CB2 1QP, Tel: +441223 333338; fax: +441223 333741;
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Email: [email protected]
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© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
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2 Abstract Background: The poor correlation between allergen‐specific‐IgE (asIgE) and clinical signs of allergy in
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helminth infected populations suggests that helminth infections could protect against allergy by uncoupling asIgE from its effector mechanisms. We investigated this hypothesis in Ugandan schoolchildren coinfected with Schistosoma mansoni and hookworm.
Methods: Skin prick test (SPT) sensitivity to house dust mite allergen (HDM) and current wheeze were assessed pre‐anthelmintic treatment. Non‐specific (anti‐IgE), helminth‐specific and HDM‐
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Results: Non‐specific‐ and helminth‐specific‐HR, and associations between helminth‐specific‐IgE and helminth‐specific‐HR increased post‐treatment. Hookworm infection appeared to modify the relationship between circulating levels of HDM‐IgE and HR: a significant positive association was
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observed among children without detectable hookworm infection but no association was observed among infected children. In addition, hookworm infection was associated with a significantly
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reduced risk of wheeze, and IgG4 to somatic adult hookworm antigen with a reduced risk of HDM‐ SPT sensitivity. There was no evidence for S. mansoni infection having a similar suppressive effect on HDM‐HR or symptoms of allergy.
Conclusions: Basophil responsiveness appears suppressed during chronic helminth infection; at least in hookworm infection, this suppression may protect against allergy.
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responses were measured pre‐ and post‐treatment.
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allergen‐specific basophil histamine release (HR), plus helminth‐ and HDM‐specific IgE and IgG4
3 Introduction
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Prevalence of allergy has increased markedly since the beginning of the 20th century[1]. Epidemiological observations suggest that this may be related to environmental and lifestyle
changes associated with urbanisation, as allergies appear more common in developed than in
developing countries[2] and even within countries there appears to be distinct division between
the burden of allergy appears also to be increasing[6].
Allergic (IgE‐mediated) disorders are usually confirmed by measuring skin prick test (SPT)
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sensitivity or allergen‐specific IgE (asIgE)[7]. However, these two measures are often poorly correlated and weakly specific for clinical phenotypes. In a large‐scale study conducted by the International Study of Asthma and Allergy in Childhood (ISAAC) group, this discordance varied
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dramatically by geography, with a strong negative correlation with gross national income per capita (GNI)[8]. Furthermore, although no overall association between asIgE and GNI was observed, there
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was a weak positive association between SPT and GNI. Similar observations have been made elsewhere[9]. One explanation could be the greater prevalence of helminth infection in lower GNI countries. Infection with helminths has similarly been associated with discordance between asIgE and SPT sensitivity[10‐12] and there is growing evidence that at least some species of helminth protect against allergy[13, 14].
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Immune responses seen in allergy and during helminth infection are markedly similar. Both
are Th2‐biased, with elevated levels of interleukin (IL)‐4, ‐5 and ‐13 cytokines, IgE and IgE‐effector
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cells, such as basophils and eosinophils. In helminthiasis, specific IgE responses are associated with immunity to infection[15‐18]; however, this immunity is only partial and appears finely balanced
between the IgE‐effector mechanisms of IgE and IgG4 down‐regulatory effects[16, 18‐21], which is
able to block IgE antigen binding and suppress IgE‐effector cells by engaging FcRIIB[22]. Regulatory
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rural and urban environments[3‐5]. Furthermore, as urbanisation increases in developing countries,
4 cells and cytokines such as Tregs, IL‐10 and TGF‐β are also up‐regulated in helminthiasis[23], together preventing allergic inflammation.
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We hypothesise that immunoregulatory responses seen in helminth infection reduce the response of IgE‐effector cells such as basophils to IgE‐mediated activation, uncoupling IgE from its
effector mechanisms, resulting in suppressed responses to both parasite and non‐parasite antigens. Here, we test this hypothesis in schoolchildren from an area co‐endemic for Schistosoma mansoni and hookworm by – 1) measuring changes in specific and non‐specific histamine release (HR) in
schistosomiasis and hookworm respectively and ‐ 2) examining associations between antigen‐
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specific‐IgE and antigen‐specific‐HR, and how these are influenced by helminth infection. We further hypothesise that the uncoupling of asIgE from its effector mechanisms has downstream effects to limit allergy; we test this by measuring house dust mite skin‐prick‐test (HDM‐SPT)
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Materials and Methods
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sensitivity and current wheeze, and how these are influenced by current helminth infection.
Study population
This study was conducted in Bwondha Village, on the shoreline of Lake Victoria, Mayuge District, Uganda. Ethical clearance was obtained from the Uganda National Council of Science and Technology. Full study details are given elsewhere[24]. Briefly, a register of all children (n=795)
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attending Bwondha Primary School was drawn up and from this, 350 children aged 7‐16 years were
selected by simple random sampling. Written informed consent was obtained from all
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parents/guardians of sampled individuals who agreed to participate. Questionnaires were administered to the most relevant parent/guardian, recording household socio‐economic characteristics (e.g. parental occupation and education, house construction, water sources, sanitation, asset ownership). Wheeze in the last 12‐months was collected by questions adapted from
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whole blood 8‐weeks after concurrent praziquantel and albendazole administration to treat
5 the ISAAC questionnaire[2]; recall bias is likely to minimal within 12‐months. All questionnaires were translated and administered in Luganda, the predominant language in Bwondha.
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Treatment, blood and stool collection
In July 2010, 350 children were treated once with albendazole (400mg) and twice with praziquantel (40mg/kg bodyweight, 1‐week apart). Venous blood samples (5ml) were collected from children
pre‐ and 8 weeks post‐treatment. Three stool samples were collected on three consecutive days,
determine treatment efficacy. Two 50mg Kato‐Katz thick‐smear slides were prepared from each
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sample and examined microscopically (within 30min of preparation for hookworm quantification). Of the 350 children recruited, 240 completed the study. Loss to follow‐up was mainly related to the transient nature of the study population.
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Parasite and environmental antigens
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Crude somatic adult hookworm antigen extract (AHW) was prepared from Ancylostoma caninum, from canines. A Puerto Rican strain of S. mansoni, maintained in outbred mice and Biomphalaria glabrata, was used for production of schistosome crude antigens. Adult worms were recovered from mice 6 weeks after infection, and parasite eggs isolated from liver tissue. Soluble worm antigen (SWA) was prepared from frozen worms, and saline‐soluble egg antigen (SEA) from frozen eggs, as
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previously described[15, 25]. House dust mite antigen (HDM) was an equal mixture of Dermatophagoides farinae and D. pteronyssinus) (Greer, Lenoir, USA).
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Skin prick testing
Skin prick testing for HDM sensitivity was conducted pre‐treatment using standard procedures[26].
Histamine was used as a positive control and saline solution as a negative control. A mean wheal diameter ≥3mm compared to the negative control was defined as a positive reaction, with the
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pre‐treatment and 5‐weeks post‐treatment, to detect treatment failure/noncompliance and
6 reading taken 15 minutes after pricking allergen onto the volar side of the forearm. HDM allergen, controls and lancets were obtained from ALK‐Abello (Horsholm, Denmark).
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Histamine release assays
Histamine‐binding glass fibre‐coated microtitre plates (RefLab ApS, Copenhagen, Denmark ) were coated with 25µl/well of antigen in triplicate, at concentrations of 7g/ml (α‐IgE, Dako, Glostrop, Denmark), 20g/ml (SEA), 20g/ml (SWA) and 7g/ml (HDM), and with histamine standards, in
were dried for 6 hours at 37˚C, then packed and sealed for use in Bwondha. 25ul/well of AHW
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antigen at 20ug/ml diluted in PIPES Buffer (RefLab ApS) was added in triplicate to plates in the field. HR assays followed a similar protocol to that described previously[27], but with the washing stage omitted, thus measuring direct HR by unwashed whole blood. Briefly, 25µl of PIPES buffer was
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added to each well of glass fibre‐coated microtitre antigen‐coated plates to dissolve antigen, followed by 25µl of unwashed whole blood. Plates were incubated for 1 hour at 37˚C to allow HR
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and then washed with distilled H2O. Plates were dried, away from direct sunlight for 24hours, then stored in the dark at room temperature before being shipped to Cambridge, UK, for histamine analysis. Histamine was measured by spectrofluorometry as described elsewhere[28], and results expressed as ng HR/mL blood.
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Antibody assays
Plasma was separated from venous blood samples and stored at ‐80°C until required. IgE and IgG4
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levels to AHW, SEA, SWA and HDM were measured by ELISA as described elsewhere[29]. Briefly,
antigen, 15l/well, was placed in 384‐well plates at saturation coating concentrations of 5g/ml (AHW), 1.2g/ml (SEA), 8g/ml (SWA) and 2g/ml (HDM) as determined by titration. 15l of sample
plasma and non‐infected plasma controls were assayed in duplicate at dilutions of 1/20 (IgE) and 1/200 (IgG4). A 3‐fold serial dilution of purified human IgG4 (Sigma‐Aldrich) or IgE myeloma
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triplicate, at 80, 50, 40, 30, and 10ug/ml, diluted in water containing 5% glycerol. Coated plates
7 (Calbiochem) was added directly to each plate to form a 14‐point standard curve, starting at 30g/ml. For schistosome and HDM assays, detection was as described in Fitzsimmons et al[29]. For
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AHW assays, detection was as described in Pinot de Moira et al[24]. For total‐IgE (tIgE) ELISAs, microplates were coated in carbonate/bicarbonate coating buffer pH9.6 with 15ul/well of mouse monoclonal anti‐human IgE antibody, clone G7‐18 at 2ug/ml (BD Pharmingen, Oxford, UK) and monoclonal mouse anti‐human biotinylated IgE (clone G7‐26) (BD Pharmingen) was used for detection. Otherwise assays were as described above.
Statistical analysis
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Infection intensity was expressed as mean egg count per gram (epg). Due to overdispersion, epg values were transformed to the logarithm, ln(epg+1) for statistical analysis. Details relating to household economic characteristics were used to construct a proxy measure of socio‐economic
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status (SES), using principle component analysis, as described previously[30, 31]. Detection thresholds for positive helminth‐specific isotype responses were calculated as the
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mean plus 3 standard deviations (SD) of non‐infected European control plasma samples, and for tIgE and anti‐HDM responses, as the mean plus 3 SDs of blank wells. Histamine readings were analysed after subtracting spontaneous HR. Due to overdispersion, antibody and histamine readings were log‐ transformed with an integer giving the best transformation, k, added/subtracted, and geometric means (GM) calculated.
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Significant changes between HR pre‐ and post‐treatment were determined using paired t‐
tests. Associations between antigen‐specific‐HR and antigen‐specific‐IgE were explored using linear
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regression analysis; the significance of any change in these associations post‐treatment was tested
using 2‐level linear regression models to allow for correlation within children, with time as “level 1”
units and child as “level 2” units, with an interaction term fitted between time and antigen‐specific‐ IgE.
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8 Two further analyses were conducted on HDM‐HR data: i) the effect modifying behaviour of hookworm infection or S. mansoni infection intensity on associations between HDM‐IgE and HDM‐
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HR were analysed using pre‐treatment data in linear regression analysis, with an interaction term between HDM‐IgE and hookworm prevalence or ln(S. mansoni epg+1); ii) change between HDM‐HR pre‐ and post‐treatment was calculated as the difference between logarithms of post‐ and pre‐
treatment HR; linear regression models were then constructed to analyse how post‐treatment HDM‐ IgE and pre‐treatment infection influenced this change, with an interaction term between HDM‐IgE
included as a binary variable for ease of interpretation and to ensure correct specification of the
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model; the high prevalence of schistosomiasis gave insufficient power to model S. mansoni as a binary variable, therefore it was included as a continuous variable.
Pre‐treatment helminth infection and antibody response effects on HDM‐SPT sensitivity and
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current wheeze was analysed using logistic regression. All regression models were adjusted for age, sex and SES; for this, age was classified into two binary groups of roughly equal size (7‐10 years, 11‐
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16 years) and SES fitted as the logarithm of the continuous variable. Multilevel models were fitted in MLwiN (Bristol University, UK); all other analyses were conducted using Stata 12.1 (StataCorp, USA). Results
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Study Characteristics
As detailed elsewhere[24], of the 350 children selected for recruitment, 240 were enrolled in the
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study and provided assent, donated at least two pre‐treatment stool samples, blood samples pre‐ and 8‐weeks post‐treatment, and complied with treatment procedures. The comparative characteristics of these 240 children and original 350 children selected are given in Table 1. S.
mansoni and hookworm infection prevalence was 93.8% (CI95%: 89.9, 96.5) and 80.4% (CI95%: 74.8,
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and hookworm prevalence or ln(S. mansoni epg+1). For both of these analyses hookworm was
9 85.2) respectively; overall GM infection intensity was 172.73 (CI95%: 131.61, 226.72) for S. mansoni and 71.75 (CI95%: 51.52, 99.90) for hookworm.
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Changes in non‐specific (α‐IgE‐induced) HR 8 weeks post anthelmintic treatment
Total IgE levels decreased significantly post‐treatment (Figure 1, p=0.04). Figure 2 shows pre‐ and
post‐treatment in vitro basophil HR following incubation of whole blood with specific antigen or α‐ IgE; P‐values indicate the level of significance for pre‐ vs. post‐treatment differences. Despite a
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Changes in hookworm‐HR 8 weeks post anthelmintic treatment
Although AHW‐IgG4 decreased significantly post‐treatment (P0.09), or for infection influencing risk of HDM‐SPT
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sensitivity (P>0.13); however, IgG4 responsiveness to AHW was associated with a reduced risk of HDM‐SPT sensitivity (ORadj=0.21, p=0.03).
Similarly to SPT‐sensitivity, prevalence of wheeze in the last 12‐months was also low, with
only 8.22% of children affected. No association was observed between HDM‐IgE or HDM‐SPT sensitivity and risk of wheeze [ORadj=1.18 (CI95%:0.58, 2.39), P=0.65 and ORadj=1.37 (0.16, 12.04),
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P=0.78 respectively], a borderline significant association was observed between HDM‐HR and risk of wheeze [ORadj=1.35 (CI95%: 0.96, 1.91), P=0.07]. Again, there was no evidence for either hookworm or
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S. mansoni infection influencing these relationships (interaction terms P>0.14); however, hookworm infection and IgG4 responsiveness to AHW were both associated with a significantly decreased risk of wheeze [ORadj=0.29 (CI95%:0.10, 0.87), P= 0.03 and ORadj=0.36 (CI95%:0.13, 1.02), P=0.05
respectively]. The protective influence of hookworm infection against wheeze appeared dose‐
dependent, with heavier hookworm infection intensities associated with lowest risk of wheeze
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had any influence on symptoms of allergy, we measured pre‐treatment SPT sensitivity to HDM and
12 [ORadj=0.80 (CI95%:0.65, 0.98), P=0.03]. There was no association between S. mansoni infection intensity and risk of wheeze [ORadj=1.05 (CI95%:0.82, 1.34)].
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Discussion
There is growing evidence that infection with some helminth species offers protection
against allergy[13, 14]. Poor correlations between as‐IgE and SPT sensitivity, and as‐IgE and clinical
uncoupling asIgE from its effector mechanisms. We investigated this hypothesis in Ugandan
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schoolchildren resident in a S. mansoni and hookworm co‐endemic area, using whole blood assays to include both cellular and humoral factors. We observed increased non‐specific and helminth‐specific HR after anthelmintic treatment, as well as stronger associations between helminth‐specific IgE and
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helminth‐specific HR. There was also evidence for hookworm infection modifying the influence of HDM‐IgE on HDM‐HR, and reducing risk of wheeze. Further, AHW‐IgG4 responsiveness was
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associated with reduced risk of HDM‐SPT sensitivity. Although average age and hookworm and S. mansoni infection prevalence were slightly higher among children completing the study, this is unlikely to have biased results; all other measured characteristics were comparable among those completing and not completing the study.
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Few studies have investigated basophil function during human helminth infection. Although basophil
competence has been demonstrated in human hookworm infection[32], ours is the first study to
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investigate treatment effects on basophil function in a hookworm‐endemic population. Basophil
function in S. mansoni‐infection, has shown similar trends towards increased HR post‐treatment. For example, adult S. mansoni‐antigen‐induced HR from basophils passively sensitised with human infection sera increased post‐treatment, and suggested that IgG4‐mediated down‐regulation occurred during active infection[28]. We also observed increases in α‐IgE‐, SEA‐ and SWA‐HR in
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signs of allergy in helminth‐infected populations[10‐12] suggests that this protection may act by
13 washed basophils from Ugandan fishermen exposed to S. mansoni[27], while Larson et al[33] reported increases in non‐specific IgE‐dependent and IgE‐independent HR in washed basophils
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following anthelmintic treatment of a small sample of Ascaris lumbricoides‐ and Trichuris trichiura‐ infected children.
We have previously observed elevated blood basophil counts in helminth‐infected
individuals using precise basophil measurements (Cahen, unpublished data), indicating that the post‐ treatment increases in measured HR to anti‐IgE, SWA‐HR and AHW‐HR observed here, truly reflect
assays to measure HR, this increase in HR could reflect changes in both cellular responsiveness (i.e.
specific IgG4).
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intracellular signal transduction) and humoral factors (i.e. circulating total and specific IgE and
Schistosomiasis treatment with praziquantel disrupts the tegument of adult worms,
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exposing otherwise sequestered allergen‐like antigens such as Schistosoma mansoni tegumental‐ allergen‐like 1 protein (SmTAL1). Hence, anti‐worm‐IgE levels often increase post‐treatment[24, 34],
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as here. Increases in SWA‐IgE could explain observed increases in SWA‐HR; however it is unlikely to explain the significant increased strength of association between SWA‐IgE and SWA‐HR. In addition, responses to schistosome egg and adult hookworm antigens are generally not boosted by treatment[24, 34, 35]. In this study, neither SEA‐IgE nor AHW‐IgE were significantly affected by treatment. However SEA‐IgG4 and AHW‐IgG4 levels fell post‐treatment, most markedly for AHW‐
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IgG4. This drop in IgG4 levels, together with increases in α‐IgE‐HR, the dramatic increases in AHW‐ HR, and the increased strength of association between helminth‐specific IgE and helminth‐specific
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HR post‐treatment, suggests a state of immunosuppresion during chronic helminth infection, which reduces basophils’ ability to respond to IgE‐mediated activation. The fact that associations between IgE and IgE‐mediated HR appear restored 8‐weeks post‐treatment, demonstrates that this helminth‐
mediated suppression is rapidly reversed upon anthelmintic treatment.
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enhanced HR rather than simply increased circulating basophil numbers. Since we used whole blood
14 Unfortunately, since schistosomiasis and hookworm were treated concurrently, it is not possible to determine whether one or both parasites are responsible for this suppression. However,
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hookworm is clearly implicated since hookworm infection significantly modified the association between HDM‐IgE and HDM‐HR. No association was observed between HDM‐IgE and HDM‐HR among hookworm‐infected children, but a strong positive association was observed among
uninfected children. Further, HDM‐HR boosted positively after treatment with increasing post‐ treatment HDM‐IgE levels in children with pre‐treatment hookworm infection, but no post‐
There is also evidence that this hookworm‐mediated suppression of basophil responsiveness
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to HDM has systemic effects. Prevalence of wheeze and HDM‐SPT sensitivity was low: only 8% and 4% of children affected respectively, despite 27% having detectable HDM‐IgE responses. This is comparable to other wheeze prevalence estimates in Africa[8, 36], but contrasts with regional
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estimates in Western Europe, North America and Oceania of 16.7‐24.2% and 8.1‐24.6% among 13‐14 and 6‐7 year olds respectively[37]. In addition, similarly to other studies[13, 38], hookworm infection
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was negatively associated with current wheeze, and AHW‐IgG4 negatively associated with both current wheeze and HDM‐SPT sensitivity.
These negative associations with AHW‐IgG4, the marked post‐treatment decrease in AHW‐
IgG4 and simultaneous increase in HR, suggest basophil suppression may be in part IgG4‐mediated. In allergen‐specific immunotherapy, increases in asIgG4 are more consistently associated with
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clinical effectiveness than changes in IgE[39], and IgG4‐containing sera from treated patients has
been shown to block basophil activation[40, 41]. Since we used an unwashed system to measure
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HR, IgG4‐mediated suppression could either be through IgG4 competing with IgE for the same epitopes, and/or through co‐engagement of low‐affinity IgG receptors FcRII, found on basophils, with the high affinity receptor FcεRI. FcRII/FcεRI co‐stimulation on human basophils has been shown to result in FcRIIB‐dependent inhibition of IgE‐induced activation[22, 42]; both with FcRII
and FcεRI co‐crosslinking the same antigen and with co‐stimulation with separate antigens[43]. The
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treatment HDM‐HR boosting was observed among uninfected children.
15 increase in non‐specific (α‐IgE‐mediated) HR post‐treatment could additionally suggest that helminth infection may also regulate basophil cell function non‐specifically, independently of specific IgG4;
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conversely however, observed increases in α‐IgE‐HR could also be related to in vitro total‐IgE mediated interference.
In conclusion, our results suggest that basophil responsiveness to IgE‐mediated activation is suppressed in S. mansoni and hookworm coinfection, but is rapidly restored by anthelmintic
treatment. Hookworm‐mediated suppression may additionally suppress HDM‐IgE‐induced HDM‐HR,
Further studies into underlying mechanisms are needed to determine if this is the case, and its
Acknowledgements
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clinical relevance to allergy management.
We are grateful for the help and cooperation of the students and staff at Bwondha Primary School
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and the parents/guardians of the children involved in this study. We also thank the field assistants, Rashid Ssalongo, Hakim Irumba, Musa Mubiru, and Musitwa, for their crucial involvement in this study. Additional gratitude extends to David Ogutu, Benjamin Tinkitina, and Moses Adriko for their invaluable assistance in the field, and to Dr Franco Falcone for is very helpful comments and suggestions.
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This study was supported by the Wellcome Trust (programme grant WT 083931/Z/07/Z and project
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grant WT 094317/Z/10/Z).
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and in turn allergy. We speculate that this basophil suppression may be in part IgG4‐mediated.
16 Footnotes
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(1) None of the authors have any conflict of interest. Per Stahl Skov is an employee of RefLab ApS, Copenhagen.
(2) This study was funded by a Wellcome Trust Programme Grant [WT 083931/Z/07/Z] and
Project Grant [WT 094317/Z/10/Z]. The funders had no role in the study design, data collection, data analysis, decision to publish, or preparation of the manuscript.
Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK.
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Tel.: +44 1223 333338; Fax: +44 1223 333741; E‐mail: [email protected]
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All reprint requests and correspondences should be addressed to: Angela Pinot de Moira,
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20. Hagan P, Blumenthal UJ, Dunn D, Simpson AJ, Wilkins HA. Human IgE, IgG4 and resistance to
reinfection with Schistosoma haematobium. Nature 1991; 349:243‐5. 21. Pinot de Moira A, Fulford AJ, Kabatereine NB, Ouma JH, Booth M, Dunne DW. Analysis of
complex patterns of human exposure and immunity to Schistosomiasis mansoni: the influence of age, sex, ethnicity and IgE. PLoS Negl Trop Dis 2010; 4.
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19 22. Cassard L, Jonsson F, Arnaud S, Daeron M. Fcgamma receptors inhibit mouse and human basophil activation. J Immunol 2012; 189:2995‐3006.
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23. McSorley HJ, Maizels RM. Helminth infections and host immune regulation. Clin Microbiol Rev 2012; 25:585‐608.
24. Pinot de Moira A, Jones FM, Wilson S, et al. Effects of treatment on IgE responses against
parasite allergen‐like proteins and immunity to reinfection in childhood schistosome and hookworm coinfections. Infect Immun 2013; 81:23‐32.
22.6‐kilodalton antigen from Schistosoma mansoni adult worms are associated with low intensities
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26. Heinzerling L, Frew AJ, Bindslev‐Jensen C, et al. Standard skin prick testing and sensitization to inhalant allergens across Europe‐‐a survey from the GALEN network. Allergy 2005; 60:1287‐300.
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27. Satti MZ, Cahen P, Skov PS, et al. Changes in IgE‐ and antigen‐dependent histamine‐release in peripheral blood of Schistosoma mansoni‐infected Ugandan fishermen after treatment with
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praziquantel. BMC Immunol 2004; 5:6.
28. Satti MZ, Ebbesen F, Vennervald B, et al. Use of a new glass microfibre histamine release method to study the modulation of the host response in human schistosomiasis mansoni. Individuals with different degrees of exposure to the disease show differing antibody biological function. Trop Med Int Health 1996; 1:655‐66.
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29. Fitzsimmons CM, Jones FM, Stearn A, et al. The Schistosoma mansoni tegumental‐allergen‐like (TAL) protein family: influence of developmental expression on human IgE responses. PLoS Negl Trop
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30. Filmer D, Pritchett L. The effect of household wealth on educational attainment: Evidence from 35 countries. Popul Dev Rev 1999; 25:85‐+. 31. Filmer D, Pritchett LH. Estimating wealth effects without expenditure data‐‐or tears: an application to educational enrollments in states of India. Demography 2001; 38:115‐32.
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25. Webster M, Fulford AJ, Braun G, et al. Human immunoglobulin E responses to a recombinant
20 32. Pritchard DI, Hooi DS, Brown A, Bockarie MJ, Caddick R, Quinnell RJ. Basophil competence during hookworm (Necator americanus) infection. Am J Trop Med Hyg 2007; 77:860‐5.
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33. Larson D, Cooper PJ, Hubner MP, et al. Helminth infection is associated with decreased basophil responsiveness in human beings. J Allergy Clin Immunol 2012; 130:270‐2.
34. Walter K, Fulford AJ, McBeath R, et al. Increased human IgE induced by killing Schistosoma
mansoni in vivo is associated with pretreatment Th2 cytokine responsiveness to worm antigens. J Immunol 2006; 177:5490‐8.
cytokine production in post‐treatment hookworm patients from an endemic area in Brazil. Clin Exp
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39. Patil SU, Shreffler WG. Immunology in the Clinic Review Series; focus on allergies: basophils as biomarkers for assessing immune modulation. Clin Exp Immunol 2011; 167:59‐66.
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41. Ejrnaes AM, Svenson M, Lund G, Larsen JN, Jacobi H. Inhibition of rBet v 1‐induced basophil histamine release with specific immunotherapy ‐induced serum immunoglobulin G: no evidence that
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35. Geiger SM, Massara CL, Bethony J, Soboslay PT, Correa‐Oliveira R. Cellular responses and
21 42. Cady CT, Powell MS, Harbeck RJ, et al. IgG antibodies produced during subcutaneous allergen immunotherapy mediate inhibition of basophil activation via a mechanism involving both
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FcgammaRII costimulation in human blood basophils. J Allergy Clin Immunol 2000; 106:337‐48.
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22 Tables
N (%) Original sample
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Table 1. Pre‐treatment characteristics of participating children
Participantsa
(n = 350)
Age (years)
7‐8
104 (29.7)
9‐10
80 (22.9)
11‐12
106 (30.3)
13‐16
60 (17.1)
40 (16.7)
Sex
Male
169 (48.3)
117 (48.8)
181 (51.7)
123 (51.3)
us 57 (23.8)
82 (34.2)
an
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SES
61 (25.4)
140 (40.0)
96 (40.0)
140 (40.0)
95 (39.6)
70 (20.0)
49 (20.4)
no wheeze
277 (92.6)
201 (91.8)
Wheeze
22 (7.36)
18 (8.2)
HDM SPT
Negative
295 (96.4)
227 (95.8)
Positive
11 (3.6)
10 (4.2)
Total‐IgE (IU/ml) [GMbc, (CI95%)]
83.58 (73.93, 94.50)
85.09 (74.74, 96.87)
Middle Upper
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Wheeze
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Female
Lower
(n = 240)
23 0.136 (0.125, 0.147)
0.138 (0.126, 0.151)
HDM‐IgE responsiveness
negative d
203 (74.1)
170 (73.0)
positive d
71 (25.9)
HDM‐IgG4 responsiveness
negative d
258 (94.2)
positive d
16 (5.8)
S. mansoni infection prevalence
319 (91.1)
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HDM‐IgE (IU/ml) [GMbc, (CI95%)]
63 (27.0)
13 (5.6)
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147.42 (116.82, 186.02)
Hookworm infection intensity [GM epg+1, (CI95%)] Other helminth infection
193 (80.4)
67.16 (50.49, 89.34)
71.75 (51.52, 99.90)
125 (35.7)
89 (37.1)e
Children completing the study, i.e. children who provided assent, donated at least two pre‐
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a
271 (77.4)
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Hookworm infection prevalence
172.73 (131.61, 226.72)
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[GM epg+1, (CI95%)]
225 (93.8)
treatment stool samples, blood samples pre‐ and 8 weeks post‐treatment, and complied with treatment procedures (n=240) b
c
GM, geometric mean
An integer, k, was added to antibody readings before calculating geometric means; k = 1.33445 (t‐
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IgE), k=0.095 (DM‐IgE) d
Detection threshold calculated as the mean+ 3 s.d. of blank wells = 0.228 (HDM‐IgE), 1.22 (HDM‐
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IgG4)
e
Specific prevalence of other helminth infections: Trichuris trichiura = 28.3%; Hymenolepis nana =
10.8%; Enterobius vermicularis = 5.4%; Ascaris lumbricoides = 4.6%
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S. mansoni infection intensity
220 (94.4)
24 Table 2. Pre‐ and post‐treatment associations between plasma levels of antigen‐specific IgE and
Adjusteda GM ratio (CI95%)b
Antigen
Pre‐treatmentc
Post‐treatmentd
t‐IgE /anti‐IgE
0.75 (0.57, 1.00)*
0.91 (0.75, 1.10)
AHW
1.05 (1.00, 1.11)
1.06 (1.02, 1.11)**
SWA
1.18 (0.92, 1.50)
1.65 (1.30, 2.10)***
histamine
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P‐valuee
release to the
0.21
respective
0.77
antigen
1.03 (0.92, 1.16)
1.16 (1.05, 1.29)**
1.10 (0.76, 1.61)
Age, sex and SES adjusted
0.89
GM, geometric mean; CI, confidence interval
c
GM increase in pre‐treatment anti‐IgE‐induced or antigen‐induced histamine release with each unit
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b
0.07
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1.15 (0.82, 1.60)
increase in pre‐treatment plasma levels of t‐IgE or IgE to the same antigen GM increase in post‐treatment anti‐IgE‐induced or antigen‐induced histamine release with each
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d
unit increase in post‐treatment plasma levels of t‐IgE or IgE to the same antigen e
Significance of any change in association between non‐specific or antigen‐specific‐HR and plasma
IgE levels pre‐ vs. post‐treatment; determined by 2‐level linear regression to allow for correlation within children, with time (pre‐ or post‐treatment) as “level 1” units and child as “level 2” units, and
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an interaction term fitted between time and antigen‐specific‐IgE *
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Angela Pinot de Moira1, Colin M. Fitzsimmons1, Frances M. Jones1, Shona Wilson1, Pierre Cahen1, Edridah Tukahebwa2, Harriet Mpairwe3, Joseph K. Mwatha4, Jeffrey M. Bethony5, Per S. Skov6, Narcis B. Kabatereine2, and David W. Dunne1
2
Vector Control Division, Ministry of Health, Kampala, PO Box 1661, Uganda
3
MRC/UVRI Uganda Research Unit on AIDS, P.O. Box 49, Entebbe, Uganda
4
Centre for Biotechnology Research and Development, Kenya Medical Research Institute, Nairobi,
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Department of Pathology, University of Cambridge, Cambridge, CB2 1QP, United Kingdom
P.O. BOX 54840 – 00200, Kenya
Department of Microbiology, Immunology, and Tropical Medicine, George Washington University,
Washington, DC 20037, USA 6
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5
RefLab ApS, DK‐2200 Copenhagen, Denmark
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Address all reprint requests or correspondence to: Angela Pinot de Moira, PhD, Department of Pathology, University of Cambridge, Cambridge, United Kingdom CB2 1QP, Tel: +441223 333338; fax: +441223 333741; Email: [email protected]
Alternative corresponding author: Prof. David Dunne, PhD, Department of Pathology, University of Cambridge, Cambridge, United Kingdom CB2 1QP, Tel: +441223 333338; fax: +441223 333741;
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Email: [email protected]
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© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
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2 Abstract Background: The poor correlation between allergen‐specific‐IgE (asIgE) and clinical signs of allergy in
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helminth infected populations suggests that helminth infections could protect against allergy by uncoupling asIgE from its effector mechanisms. We investigated this hypothesis in Ugandan schoolchildren coinfected with Schistosoma mansoni and hookworm.
Methods: Skin prick test (SPT) sensitivity to house dust mite allergen (HDM) and current wheeze were assessed pre‐anthelmintic treatment. Non‐specific (anti‐IgE), helminth‐specific and HDM‐
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Results: Non‐specific‐ and helminth‐specific‐HR, and associations between helminth‐specific‐IgE and helminth‐specific‐HR increased post‐treatment. Hookworm infection appeared to modify the relationship between circulating levels of HDM‐IgE and HR: a significant positive association was
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observed among children without detectable hookworm infection but no association was observed among infected children. In addition, hookworm infection was associated with a significantly
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reduced risk of wheeze, and IgG4 to somatic adult hookworm antigen with a reduced risk of HDM‐ SPT sensitivity. There was no evidence for S. mansoni infection having a similar suppressive effect on HDM‐HR or symptoms of allergy.
Conclusions: Basophil responsiveness appears suppressed during chronic helminth infection; at least in hookworm infection, this suppression may protect against allergy.
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responses were measured pre‐ and post‐treatment.
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allergen‐specific basophil histamine release (HR), plus helminth‐ and HDM‐specific IgE and IgG4
3 Introduction
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Prevalence of allergy has increased markedly since the beginning of the 20th century[1]. Epidemiological observations suggest that this may be related to environmental and lifestyle
changes associated with urbanisation, as allergies appear more common in developed than in
developing countries[2] and even within countries there appears to be distinct division between
the burden of allergy appears also to be increasing[6].
Allergic (IgE‐mediated) disorders are usually confirmed by measuring skin prick test (SPT)
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sensitivity or allergen‐specific IgE (asIgE)[7]. However, these two measures are often poorly correlated and weakly specific for clinical phenotypes. In a large‐scale study conducted by the International Study of Asthma and Allergy in Childhood (ISAAC) group, this discordance varied
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dramatically by geography, with a strong negative correlation with gross national income per capita (GNI)[8]. Furthermore, although no overall association between asIgE and GNI was observed, there
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was a weak positive association between SPT and GNI. Similar observations have been made elsewhere[9]. One explanation could be the greater prevalence of helminth infection in lower GNI countries. Infection with helminths has similarly been associated with discordance between asIgE and SPT sensitivity[10‐12] and there is growing evidence that at least some species of helminth protect against allergy[13, 14].
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Immune responses seen in allergy and during helminth infection are markedly similar. Both
are Th2‐biased, with elevated levels of interleukin (IL)‐4, ‐5 and ‐13 cytokines, IgE and IgE‐effector
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cells, such as basophils and eosinophils. In helminthiasis, specific IgE responses are associated with immunity to infection[15‐18]; however, this immunity is only partial and appears finely balanced
between the IgE‐effector mechanisms of IgE and IgG4 down‐regulatory effects[16, 18‐21], which is
able to block IgE antigen binding and suppress IgE‐effector cells by engaging FcRIIB[22]. Regulatory
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rural and urban environments[3‐5]. Furthermore, as urbanisation increases in developing countries,
4 cells and cytokines such as Tregs, IL‐10 and TGF‐β are also up‐regulated in helminthiasis[23], together preventing allergic inflammation.
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We hypothesise that immunoregulatory responses seen in helminth infection reduce the response of IgE‐effector cells such as basophils to IgE‐mediated activation, uncoupling IgE from its
effector mechanisms, resulting in suppressed responses to both parasite and non‐parasite antigens. Here, we test this hypothesis in schoolchildren from an area co‐endemic for Schistosoma mansoni and hookworm by – 1) measuring changes in specific and non‐specific histamine release (HR) in
schistosomiasis and hookworm respectively and ‐ 2) examining associations between antigen‐
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specific‐IgE and antigen‐specific‐HR, and how these are influenced by helminth infection. We further hypothesise that the uncoupling of asIgE from its effector mechanisms has downstream effects to limit allergy; we test this by measuring house dust mite skin‐prick‐test (HDM‐SPT)
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Materials and Methods
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sensitivity and current wheeze, and how these are influenced by current helminth infection.
Study population
This study was conducted in Bwondha Village, on the shoreline of Lake Victoria, Mayuge District, Uganda. Ethical clearance was obtained from the Uganda National Council of Science and Technology. Full study details are given elsewhere[24]. Briefly, a register of all children (n=795)
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attending Bwondha Primary School was drawn up and from this, 350 children aged 7‐16 years were
selected by simple random sampling. Written informed consent was obtained from all
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parents/guardians of sampled individuals who agreed to participate. Questionnaires were administered to the most relevant parent/guardian, recording household socio‐economic characteristics (e.g. parental occupation and education, house construction, water sources, sanitation, asset ownership). Wheeze in the last 12‐months was collected by questions adapted from
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whole blood 8‐weeks after concurrent praziquantel and albendazole administration to treat
5 the ISAAC questionnaire[2]; recall bias is likely to minimal within 12‐months. All questionnaires were translated and administered in Luganda, the predominant language in Bwondha.
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Treatment, blood and stool collection
In July 2010, 350 children were treated once with albendazole (400mg) and twice with praziquantel (40mg/kg bodyweight, 1‐week apart). Venous blood samples (5ml) were collected from children
pre‐ and 8 weeks post‐treatment. Three stool samples were collected on three consecutive days,
determine treatment efficacy. Two 50mg Kato‐Katz thick‐smear slides were prepared from each
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sample and examined microscopically (within 30min of preparation for hookworm quantification). Of the 350 children recruited, 240 completed the study. Loss to follow‐up was mainly related to the transient nature of the study population.
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Parasite and environmental antigens
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Crude somatic adult hookworm antigen extract (AHW) was prepared from Ancylostoma caninum, from canines. A Puerto Rican strain of S. mansoni, maintained in outbred mice and Biomphalaria glabrata, was used for production of schistosome crude antigens. Adult worms were recovered from mice 6 weeks after infection, and parasite eggs isolated from liver tissue. Soluble worm antigen (SWA) was prepared from frozen worms, and saline‐soluble egg antigen (SEA) from frozen eggs, as
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previously described[15, 25]. House dust mite antigen (HDM) was an equal mixture of Dermatophagoides farinae and D. pteronyssinus) (Greer, Lenoir, USA).
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Skin prick testing
Skin prick testing for HDM sensitivity was conducted pre‐treatment using standard procedures[26].
Histamine was used as a positive control and saline solution as a negative control. A mean wheal diameter ≥3mm compared to the negative control was defined as a positive reaction, with the
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pre‐treatment and 5‐weeks post‐treatment, to detect treatment failure/noncompliance and
6 reading taken 15 minutes after pricking allergen onto the volar side of the forearm. HDM allergen, controls and lancets were obtained from ALK‐Abello (Horsholm, Denmark).
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Histamine release assays
Histamine‐binding glass fibre‐coated microtitre plates (RefLab ApS, Copenhagen, Denmark ) were coated with 25µl/well of antigen in triplicate, at concentrations of 7g/ml (α‐IgE, Dako, Glostrop, Denmark), 20g/ml (SEA), 20g/ml (SWA) and 7g/ml (HDM), and with histamine standards, in
were dried for 6 hours at 37˚C, then packed and sealed for use in Bwondha. 25ul/well of AHW
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antigen at 20ug/ml diluted in PIPES Buffer (RefLab ApS) was added in triplicate to plates in the field. HR assays followed a similar protocol to that described previously[27], but with the washing stage omitted, thus measuring direct HR by unwashed whole blood. Briefly, 25µl of PIPES buffer was
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added to each well of glass fibre‐coated microtitre antigen‐coated plates to dissolve antigen, followed by 25µl of unwashed whole blood. Plates were incubated for 1 hour at 37˚C to allow HR
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and then washed with distilled H2O. Plates were dried, away from direct sunlight for 24hours, then stored in the dark at room temperature before being shipped to Cambridge, UK, for histamine analysis. Histamine was measured by spectrofluorometry as described elsewhere[28], and results expressed as ng HR/mL blood.
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Antibody assays
Plasma was separated from venous blood samples and stored at ‐80°C until required. IgE and IgG4
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levels to AHW, SEA, SWA and HDM were measured by ELISA as described elsewhere[29]. Briefly,
antigen, 15l/well, was placed in 384‐well plates at saturation coating concentrations of 5g/ml (AHW), 1.2g/ml (SEA), 8g/ml (SWA) and 2g/ml (HDM) as determined by titration. 15l of sample
plasma and non‐infected plasma controls were assayed in duplicate at dilutions of 1/20 (IgE) and 1/200 (IgG4). A 3‐fold serial dilution of purified human IgG4 (Sigma‐Aldrich) or IgE myeloma
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triplicate, at 80, 50, 40, 30, and 10ug/ml, diluted in water containing 5% glycerol. Coated plates
7 (Calbiochem) was added directly to each plate to form a 14‐point standard curve, starting at 30g/ml. For schistosome and HDM assays, detection was as described in Fitzsimmons et al[29]. For
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AHW assays, detection was as described in Pinot de Moira et al[24]. For total‐IgE (tIgE) ELISAs, microplates were coated in carbonate/bicarbonate coating buffer pH9.6 with 15ul/well of mouse monoclonal anti‐human IgE antibody, clone G7‐18 at 2ug/ml (BD Pharmingen, Oxford, UK) and monoclonal mouse anti‐human biotinylated IgE (clone G7‐26) (BD Pharmingen) was used for detection. Otherwise assays were as described above.
Statistical analysis
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Infection intensity was expressed as mean egg count per gram (epg). Due to overdispersion, epg values were transformed to the logarithm, ln(epg+1) for statistical analysis. Details relating to household economic characteristics were used to construct a proxy measure of socio‐economic
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status (SES), using principle component analysis, as described previously[30, 31]. Detection thresholds for positive helminth‐specific isotype responses were calculated as the
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mean plus 3 standard deviations (SD) of non‐infected European control plasma samples, and for tIgE and anti‐HDM responses, as the mean plus 3 SDs of blank wells. Histamine readings were analysed after subtracting spontaneous HR. Due to overdispersion, antibody and histamine readings were log‐ transformed with an integer giving the best transformation, k, added/subtracted, and geometric means (GM) calculated.
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Significant changes between HR pre‐ and post‐treatment were determined using paired t‐
tests. Associations between antigen‐specific‐HR and antigen‐specific‐IgE were explored using linear
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regression analysis; the significance of any change in these associations post‐treatment was tested
using 2‐level linear regression models to allow for correlation within children, with time as “level 1”
units and child as “level 2” units, with an interaction term fitted between time and antigen‐specific‐ IgE.
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8 Two further analyses were conducted on HDM‐HR data: i) the effect modifying behaviour of hookworm infection or S. mansoni infection intensity on associations between HDM‐IgE and HDM‐
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HR were analysed using pre‐treatment data in linear regression analysis, with an interaction term between HDM‐IgE and hookworm prevalence or ln(S. mansoni epg+1); ii) change between HDM‐HR pre‐ and post‐treatment was calculated as the difference between logarithms of post‐ and pre‐
treatment HR; linear regression models were then constructed to analyse how post‐treatment HDM‐ IgE and pre‐treatment infection influenced this change, with an interaction term between HDM‐IgE
included as a binary variable for ease of interpretation and to ensure correct specification of the
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model; the high prevalence of schistosomiasis gave insufficient power to model S. mansoni as a binary variable, therefore it was included as a continuous variable.
Pre‐treatment helminth infection and antibody response effects on HDM‐SPT sensitivity and
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current wheeze was analysed using logistic regression. All regression models were adjusted for age, sex and SES; for this, age was classified into two binary groups of roughly equal size (7‐10 years, 11‐
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16 years) and SES fitted as the logarithm of the continuous variable. Multilevel models were fitted in MLwiN (Bristol University, UK); all other analyses were conducted using Stata 12.1 (StataCorp, USA). Results
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Study Characteristics
As detailed elsewhere[24], of the 350 children selected for recruitment, 240 were enrolled in the
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study and provided assent, donated at least two pre‐treatment stool samples, blood samples pre‐ and 8‐weeks post‐treatment, and complied with treatment procedures. The comparative characteristics of these 240 children and original 350 children selected are given in Table 1. S.
mansoni and hookworm infection prevalence was 93.8% (CI95%: 89.9, 96.5) and 80.4% (CI95%: 74.8,
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and hookworm prevalence or ln(S. mansoni epg+1). For both of these analyses hookworm was
9 85.2) respectively; overall GM infection intensity was 172.73 (CI95%: 131.61, 226.72) for S. mansoni and 71.75 (CI95%: 51.52, 99.90) for hookworm.
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Changes in non‐specific (α‐IgE‐induced) HR 8 weeks post anthelmintic treatment
Total IgE levels decreased significantly post‐treatment (Figure 1, p=0.04). Figure 2 shows pre‐ and
post‐treatment in vitro basophil HR following incubation of whole blood with specific antigen or α‐ IgE; P‐values indicate the level of significance for pre‐ vs. post‐treatment differences. Despite a
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Changes in hookworm‐HR 8 weeks post anthelmintic treatment
Although AHW‐IgG4 decreased significantly post‐treatment (P0.09), or for infection influencing risk of HDM‐SPT
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sensitivity (P>0.13); however, IgG4 responsiveness to AHW was associated with a reduced risk of HDM‐SPT sensitivity (ORadj=0.21, p=0.03).
Similarly to SPT‐sensitivity, prevalence of wheeze in the last 12‐months was also low, with
only 8.22% of children affected. No association was observed between HDM‐IgE or HDM‐SPT sensitivity and risk of wheeze [ORadj=1.18 (CI95%:0.58, 2.39), P=0.65 and ORadj=1.37 (0.16, 12.04),
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P=0.78 respectively], a borderline significant association was observed between HDM‐HR and risk of wheeze [ORadj=1.35 (CI95%: 0.96, 1.91), P=0.07]. Again, there was no evidence for either hookworm or
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S. mansoni infection influencing these relationships (interaction terms P>0.14); however, hookworm infection and IgG4 responsiveness to AHW were both associated with a significantly decreased risk of wheeze [ORadj=0.29 (CI95%:0.10, 0.87), P= 0.03 and ORadj=0.36 (CI95%:0.13, 1.02), P=0.05
respectively]. The protective influence of hookworm infection against wheeze appeared dose‐
dependent, with heavier hookworm infection intensities associated with lowest risk of wheeze
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had any influence on symptoms of allergy, we measured pre‐treatment SPT sensitivity to HDM and
12 [ORadj=0.80 (CI95%:0.65, 0.98), P=0.03]. There was no association between S. mansoni infection intensity and risk of wheeze [ORadj=1.05 (CI95%:0.82, 1.34)].
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Discussion
There is growing evidence that infection with some helminth species offers protection
against allergy[13, 14]. Poor correlations between as‐IgE and SPT sensitivity, and as‐IgE and clinical
uncoupling asIgE from its effector mechanisms. We investigated this hypothesis in Ugandan
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schoolchildren resident in a S. mansoni and hookworm co‐endemic area, using whole blood assays to include both cellular and humoral factors. We observed increased non‐specific and helminth‐specific HR after anthelmintic treatment, as well as stronger associations between helminth‐specific IgE and
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helminth‐specific HR. There was also evidence for hookworm infection modifying the influence of HDM‐IgE on HDM‐HR, and reducing risk of wheeze. Further, AHW‐IgG4 responsiveness was
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associated with reduced risk of HDM‐SPT sensitivity. Although average age and hookworm and S. mansoni infection prevalence were slightly higher among children completing the study, this is unlikely to have biased results; all other measured characteristics were comparable among those completing and not completing the study.
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Few studies have investigated basophil function during human helminth infection. Although basophil
competence has been demonstrated in human hookworm infection[32], ours is the first study to
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investigate treatment effects on basophil function in a hookworm‐endemic population. Basophil
function in S. mansoni‐infection, has shown similar trends towards increased HR post‐treatment. For example, adult S. mansoni‐antigen‐induced HR from basophils passively sensitised with human infection sera increased post‐treatment, and suggested that IgG4‐mediated down‐regulation occurred during active infection[28]. We also observed increases in α‐IgE‐, SEA‐ and SWA‐HR in
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signs of allergy in helminth‐infected populations[10‐12] suggests that this protection may act by
13 washed basophils from Ugandan fishermen exposed to S. mansoni[27], while Larson et al[33] reported increases in non‐specific IgE‐dependent and IgE‐independent HR in washed basophils
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following anthelmintic treatment of a small sample of Ascaris lumbricoides‐ and Trichuris trichiura‐ infected children.
We have previously observed elevated blood basophil counts in helminth‐infected
individuals using precise basophil measurements (Cahen, unpublished data), indicating that the post‐ treatment increases in measured HR to anti‐IgE, SWA‐HR and AHW‐HR observed here, truly reflect
assays to measure HR, this increase in HR could reflect changes in both cellular responsiveness (i.e.
specific IgG4).
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intracellular signal transduction) and humoral factors (i.e. circulating total and specific IgE and
Schistosomiasis treatment with praziquantel disrupts the tegument of adult worms,
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exposing otherwise sequestered allergen‐like antigens such as Schistosoma mansoni tegumental‐ allergen‐like 1 protein (SmTAL1). Hence, anti‐worm‐IgE levels often increase post‐treatment[24, 34],
pt ed
as here. Increases in SWA‐IgE could explain observed increases in SWA‐HR; however it is unlikely to explain the significant increased strength of association between SWA‐IgE and SWA‐HR. In addition, responses to schistosome egg and adult hookworm antigens are generally not boosted by treatment[24, 34, 35]. In this study, neither SEA‐IgE nor AHW‐IgE were significantly affected by treatment. However SEA‐IgG4 and AHW‐IgG4 levels fell post‐treatment, most markedly for AHW‐
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IgG4. This drop in IgG4 levels, together with increases in α‐IgE‐HR, the dramatic increases in AHW‐ HR, and the increased strength of association between helminth‐specific IgE and helminth‐specific
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HR post‐treatment, suggests a state of immunosuppresion during chronic helminth infection, which reduces basophils’ ability to respond to IgE‐mediated activation. The fact that associations between IgE and IgE‐mediated HR appear restored 8‐weeks post‐treatment, demonstrates that this helminth‐
mediated suppression is rapidly reversed upon anthelmintic treatment.
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enhanced HR rather than simply increased circulating basophil numbers. Since we used whole blood
14 Unfortunately, since schistosomiasis and hookworm were treated concurrently, it is not possible to determine whether one or both parasites are responsible for this suppression. However,
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hookworm is clearly implicated since hookworm infection significantly modified the association between HDM‐IgE and HDM‐HR. No association was observed between HDM‐IgE and HDM‐HR among hookworm‐infected children, but a strong positive association was observed among
uninfected children. Further, HDM‐HR boosted positively after treatment with increasing post‐ treatment HDM‐IgE levels in children with pre‐treatment hookworm infection, but no post‐
There is also evidence that this hookworm‐mediated suppression of basophil responsiveness
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to HDM has systemic effects. Prevalence of wheeze and HDM‐SPT sensitivity was low: only 8% and 4% of children affected respectively, despite 27% having detectable HDM‐IgE responses. This is comparable to other wheeze prevalence estimates in Africa[8, 36], but contrasts with regional
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estimates in Western Europe, North America and Oceania of 16.7‐24.2% and 8.1‐24.6% among 13‐14 and 6‐7 year olds respectively[37]. In addition, similarly to other studies[13, 38], hookworm infection
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was negatively associated with current wheeze, and AHW‐IgG4 negatively associated with both current wheeze and HDM‐SPT sensitivity.
These negative associations with AHW‐IgG4, the marked post‐treatment decrease in AHW‐
IgG4 and simultaneous increase in HR, suggest basophil suppression may be in part IgG4‐mediated. In allergen‐specific immunotherapy, increases in asIgG4 are more consistently associated with
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clinical effectiveness than changes in IgE[39], and IgG4‐containing sera from treated patients has
been shown to block basophil activation[40, 41]. Since we used an unwashed system to measure
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HR, IgG4‐mediated suppression could either be through IgG4 competing with IgE for the same epitopes, and/or through co‐engagement of low‐affinity IgG receptors FcRII, found on basophils, with the high affinity receptor FcεRI. FcRII/FcεRI co‐stimulation on human basophils has been shown to result in FcRIIB‐dependent inhibition of IgE‐induced activation[22, 42]; both with FcRII
and FcεRI co‐crosslinking the same antigen and with co‐stimulation with separate antigens[43]. The
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treatment HDM‐HR boosting was observed among uninfected children.
15 increase in non‐specific (α‐IgE‐mediated) HR post‐treatment could additionally suggest that helminth infection may also regulate basophil cell function non‐specifically, independently of specific IgG4;
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conversely however, observed increases in α‐IgE‐HR could also be related to in vitro total‐IgE mediated interference.
In conclusion, our results suggest that basophil responsiveness to IgE‐mediated activation is suppressed in S. mansoni and hookworm coinfection, but is rapidly restored by anthelmintic
treatment. Hookworm‐mediated suppression may additionally suppress HDM‐IgE‐induced HDM‐HR,
Further studies into underlying mechanisms are needed to determine if this is the case, and its
Acknowledgements
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clinical relevance to allergy management.
We are grateful for the help and cooperation of the students and staff at Bwondha Primary School
pt ed
and the parents/guardians of the children involved in this study. We also thank the field assistants, Rashid Ssalongo, Hakim Irumba, Musa Mubiru, and Musitwa, for their crucial involvement in this study. Additional gratitude extends to David Ogutu, Benjamin Tinkitina, and Moses Adriko for their invaluable assistance in the field, and to Dr Franco Falcone for is very helpful comments and suggestions.
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This study was supported by the Wellcome Trust (programme grant WT 083931/Z/07/Z and project
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grant WT 094317/Z/10/Z).
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and in turn allergy. We speculate that this basophil suppression may be in part IgG4‐mediated.
16 Footnotes
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(1) None of the authors have any conflict of interest. Per Stahl Skov is an employee of RefLab ApS, Copenhagen.
(2) This study was funded by a Wellcome Trust Programme Grant [WT 083931/Z/07/Z] and
Project Grant [WT 094317/Z/10/Z]. The funders had no role in the study design, data collection, data analysis, decision to publish, or preparation of the manuscript.
Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK.
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Tel.: +44 1223 333338; Fax: +44 1223 333741; E‐mail: [email protected]
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All reprint requests and correspondences should be addressed to: Angela Pinot de Moira,
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22 Tables
N (%) Original sample
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Table 1. Pre‐treatment characteristics of participating children
Participantsa
(n = 350)
Age (years)
7‐8
104 (29.7)
9‐10
80 (22.9)
11‐12
106 (30.3)
13‐16
60 (17.1)
40 (16.7)
Sex
Male
169 (48.3)
117 (48.8)
181 (51.7)
123 (51.3)
us 57 (23.8)
82 (34.2)
an
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SES
61 (25.4)
140 (40.0)
96 (40.0)
140 (40.0)
95 (39.6)
70 (20.0)
49 (20.4)
no wheeze
277 (92.6)
201 (91.8)
Wheeze
22 (7.36)
18 (8.2)
HDM SPT
Negative
295 (96.4)
227 (95.8)
Positive
11 (3.6)
10 (4.2)
Total‐IgE (IU/ml) [GMbc, (CI95%)]
83.58 (73.93, 94.50)
85.09 (74.74, 96.87)
Middle Upper
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Wheeze
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Female
Lower
(n = 240)
23 0.136 (0.125, 0.147)
0.138 (0.126, 0.151)
HDM‐IgE responsiveness
negative d
203 (74.1)
170 (73.0)
positive d
71 (25.9)
HDM‐IgG4 responsiveness
negative d
258 (94.2)
positive d
16 (5.8)
S. mansoni infection prevalence
319 (91.1)
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HDM‐IgE (IU/ml) [GMbc, (CI95%)]
63 (27.0)
13 (5.6)
us
147.42 (116.82, 186.02)
Hookworm infection intensity [GM epg+1, (CI95%)] Other helminth infection
193 (80.4)
67.16 (50.49, 89.34)
71.75 (51.52, 99.90)
125 (35.7)
89 (37.1)e
Children completing the study, i.e. children who provided assent, donated at least two pre‐
pt ed
a
271 (77.4)
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Hookworm infection prevalence
172.73 (131.61, 226.72)
an
[GM epg+1, (CI95%)]
225 (93.8)
treatment stool samples, blood samples pre‐ and 8 weeks post‐treatment, and complied with treatment procedures (n=240) b
c
GM, geometric mean
An integer, k, was added to antibody readings before calculating geometric means; k = 1.33445 (t‐
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IgE), k=0.095 (DM‐IgE) d
Detection threshold calculated as the mean+ 3 s.d. of blank wells = 0.228 (HDM‐IgE), 1.22 (HDM‐
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IgG4)
e
Specific prevalence of other helminth infections: Trichuris trichiura = 28.3%; Hymenolepis nana =
10.8%; Enterobius vermicularis = 5.4%; Ascaris lumbricoides = 4.6%
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S. mansoni infection intensity
220 (94.4)
24 Table 2. Pre‐ and post‐treatment associations between plasma levels of antigen‐specific IgE and
Adjusteda GM ratio (CI95%)b
Antigen
Pre‐treatmentc
Post‐treatmentd
t‐IgE /anti‐IgE
0.75 (0.57, 1.00)*
0.91 (0.75, 1.10)
AHW
1.05 (1.00, 1.11)
1.06 (1.02, 1.11)**
SWA
1.18 (0.92, 1.50)
1.65 (1.30, 2.10)***
histamine
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P‐valuee
release to the
0.21
respective
0.77
antigen
1.03 (0.92, 1.16)
1.16 (1.05, 1.29)**
1.10 (0.76, 1.61)
Age, sex and SES adjusted
0.89
GM, geometric mean; CI, confidence interval
c
GM increase in pre‐treatment anti‐IgE‐induced or antigen‐induced histamine release with each unit
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b
0.07
an
1.15 (0.82, 1.60)
increase in pre‐treatment plasma levels of t‐IgE or IgE to the same antigen GM increase in post‐treatment anti‐IgE‐induced or antigen‐induced histamine release with each
pt ed
d
unit increase in post‐treatment plasma levels of t‐IgE or IgE to the same antigen e
Significance of any change in association between non‐specific or antigen‐specific‐HR and plasma
IgE levels pre‐ vs. post‐treatment; determined by 2‐level linear regression to allow for correlation within children, with time (pre‐ or post‐treatment) as “level 1” units and child as “level 2” units, and
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an interaction term fitted between time and antigen‐specific‐IgE *
